[Histonet] GS isolectin questions

[Jacqui Detmar]

Hi Tyrone.  I use that same lectin to stain mouse placental endothelial
cells.  I make a 1 mg/mL solution using PBS and then dilute it to 50
ug/mL (0.05 mg/mL), which is a 1:20 dilution.  You could probably try a
lower concentration, but I found for my mouse placentae that if I went
lower, the stain became fainter.  Good luck!

Jacqui

Jacqui Detmar, Post-doctoral Fellow
Samuel Lunenfeld Research Institute, 
Mount Sinai Hospital
25 Orde Street, room 6-1001 AJ,
Toronto, ON, Canada
M5T 3H7
 
phone:   416-586-4800 x5607
fax:        416-586-8588
email:     detmar <@t> mshri.on.ca
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Tyrone
Genade
Sent: Friday, July 04, 2008 3:48 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] GS isolectin questions

Hello,

I'm new to the list and the field of histology (busy startinga PhD).

I have a question regarding GS BS-I Isolectin B4 (Sigma). This was the
lectin used in the article by Kim et al (Neurobiology of Aging
25:491--500, 2004) for selective staining of CNS microglia. They state
that it was used at a dilution of 1:50 which is amazingly helpful in
light of the fact that no starting concentration is stated. I have
been able to contact the corresponding author who gave me the email of
the author who did the lectin work but no response...

I have purchased 1 mg of the GS isolectin which has now arrived in
powdered form. The recommendation by Sigma is to dissovle it in to
0.9% NaCl. Can anyone suggest a starting concentration from which I
can make the 1:50 dilutions or better, suggest a good working solution
concentration.

I will be staining retinal sections of fish, not mammals, so anyone
with advise pertinent to fish histology would be appreciated.

I understand that as negative control I will have treat control
sections with a lactose solution. Can anyone volunteer more
information regarding control protocols? Also, I may want to counter
stain with an antibody in frozen sections. Would the BSA blocking
solution interfer with the lectin binding. We had previously been
using tomato lectin with dismal results (it stuck to everything!).

Many thanks

Tyrone

-- 
Tyrone Genade
http://tgenade.freeshell.org
email: tgenade <@t> freeshell.org
tel: +27-84-632-1925 (c)
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"For there is one God, and there is one mediator between God and
men, the man Christ Jesus, who gave himself as a ransom for all."
        1 Timothy 2:5-6

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